Effective isolation of nucleic acids from biological samples (e.g. cultured cells, tissue, viruses) is an essential prerequisite for efficient downstream amplification, detection, and quantification of specific genetic sequences via quantitative polymerase chain reaction (qPCR). The extraction process requires lysing the cells with harsh extraction reagents, such as detergents or enzymes, thereby resulting in a mixture of nucleic acids, cellular debris and extraction reagents. The nucleic acids are then separated/purified from the cellular debris and extraction reagents using a variety of techniques (e.g. organic solvent extraction, chromatography, centrifugation, dialysis). These techniques can be very time-consuming, tedious, and often require multiple washing steps. By way of example, commercially-available nucleic acid isolation kits require approximately 15 minutes to over one hour to complete, largely due to the multiple washing steps required to sufficiently separate the nucleic acids from the cellular debris and extraction reagents. Consequently, it has been suggested that as much as 15% of all molecular biology research time is devoted to purification.
In view of the foregoing, various attempts have been made to reduce the time associated with isolating nucleic acids from a biological sample. By way of example, Kelso, United States Patent Application No. 20090246782 discloses a system, device, and method for performing biological reactions. More specifically, the system contemplates placing a sample in a first chamber. The first chamber includes first processing reagents to generate a processed sample. The processed sample is moved through a water and alcohol immiscible, hydrophobic, or lipophilic barrier to a second chamber. The processed sample is treated in said second chamber with second processing reagents to generate a further processed sample.
While functional for its intended purpose, the system disclosed in the '782 application has certain limitations. For example, the reagents and immiscible phase of the system disclosed in the '782 application must be confined within corresponding chambers. As a result, the system requires the use of an external pump or two-axis magnet to move the processed sample between the chambers. It can be appreciated that the use of an external pump may have undesired effects on the sample. Alternatively, the use of a two-axis magnet may add unwanted cost and complexity to the system. In addition, the use of a plurality of chambers to isolate the nucleic acids from a biological sample may limit the throughput of the system.
Therefore, it is a primary object and feature of the present invention to provide a method of extracting and purifying a fraction from cultured cells, tissue samples and other biological materials.
It is a further object and feature of the present invention to provide a method of extracting and purifying a fraction from cultured cells, tissue samples and other biological materials that is simpler and more efficient than prior methods.
It is a still further object and feature of the present invention to provide a method of extracting and purifying a fraction from cultured cells, tissue samples and other biological materials that has higher throughput than prior methods.
In accordance with the present invention, a method is provided for facilitating extraction of a fraction from a biological sample. The biological sample includes non-desired material and a fraction-bound solid phase substrate. The method includes the steps of capturing the fraction-bound solid phase substrate and bringing an isolation buffer and the fraction-bound solid phase substrate into contact to purify the captured fraction-bound solid phase substrate.
The method may also include the step of sequentially drawing the isolation buffer and the biological sample into a barrel of a syringe. The step of bringing the isolation buffer and the fraction-bound solid phase substrate into contact includes the step of sequentially urging the biological sample and the isolation buffer from the barrel of the syringe. Alternatively, the step of bringing an isolation buffer and the fraction-bound solid phase substrate into contact may include the additional steps adhering the isolation buffer to a surface and bringing the surface into contact with the fraction-bound solid phase substrate. In order to bring the surface into contact with the fraction-bound solid phase substrate, the surface may be slid across the fraction-bound solid phase substrate.
Further, it is contemplated to position the captured fraction-bound solid phase substrate in an air channel prior to the step of bringing the isolation buffer and the fraction-bound solid phase substrate into contact. In order to position the captured fraction-bound solid phase substrate in the air channel, the biological sample may be allowed to evaporate. Alternatively, the captured fraction-bound solid phase substrate fraction may be drawn into the air channel with a magnetic force.
In accordance with a further aspect of the present invention, a method is provided for facilitating extraction of a fraction from a biological sample. The biological sample including non-desired material and a fraction-bound solid phase substrate. The method includes the step of capturing the fraction-bound solid phase substrate with a magnetic force. The captured fraction-bound solid phase substrate is purified with an isolation buffer.
The method may include the additional step of sequentially drawing the isolation buffer and the biological sample into a barrel of a syringe prior to capturing the fraction-bound solid phase substrate. The captured fraction-bound solid phase substrate is purified by sequentially urging the biological sample and the isolation buffer from the barrel of the syringe. Alternatively, the captured fraction-bound solid phase substrate may be purified by adhering the isolation buffer to a surface and bringing the surface into contact with the fraction-bound solid phase substrate. For example, the surface may be slid across the fraction-bound solid phase substrate.
The captured fraction-bound solid phase substrate may be positioned in an air channel prior to the step of purifying the captured fraction-bound solid phase substrate fraction. The captured fraction-bound solid phase substrate may be positioned in the air channel by allowing the biological sample to evaporate. Alternatively, the captured fraction-bound solid phase substrate may be drawn into the air channel with a magnetic force.
In accordance with a still aspect of the present invention, a method is provided for facilitating extraction of a fraction from a biological sample. The biological sample includes non-desired material and a fraction-bound solid phase substrate. The method includes the steps of capturing the fraction-bound solid phase substrate with a magnetic force and moving an isolation buffer into contact with the captured fraction-bound solid phase substrate.
The method may include the additional step of sequentially drawing the isolation buffer and the biological sample into a barrel of a syringe prior to capturing the fraction-bound solid phase substrate. The isolation buffer may be moved by sequentially urging the biological sample and the isolation buffer from the barrel of the syringe. Alternatively, the isolation buffer may be moved by adhering the isolation buffer to a surface and bringing the surface into contact with the fraction-bound solid phase substrate. By way of example, the surface may be slid across the fraction-bound solid phase substrate.
Prior to moving the isolation buffer into contact with the fraction-bound solid phase substrate, the captured fraction-bound solid phase substrate may be positioned in an air. In order to position the captured fraction-bound solid phase substrate in the air channel, the biological sample may be allowed to evaporate. Alternatively, the captured fraction-bound solid phase substrate fraction may be drawn into the air channel with a magnetic force.